Envision flex high ph kit

BTBR^ob/ob and wild type mice kidneys were fixed with formalin (10%) and sectioned at 3–4 µm thick. IHC reaction was performed with EnVision FLEX High pH kit (#K8000—DAKO, Carpinteria, CA, USA). Scoring for WT-1⁺ cells was carried out by counting the number of positive nuclei in 25 glomeruli randomly chosen in kidney cortexes and outer medulla sections using 10× magnification and after applying a rabbit polyclonal WT-1 antibody (anti-WT1; #sc-192—Santa Cruz, Dallas, TX, USA). Data from all fields and all kidneys were pooled to obtain the number of WT-1⁺ podocytes per glomerular cross-section. For autophagy induction, quantification of LC3 (anti-LC3; #4445S—Cell Signaling, Danvers, MA, USA) was performed analyzing cortex and medulla regions. For apoptosis pathway analysis, we evaluated caspase-3 and cleaved caspase-3 (#9662; #9661—Cell Signaling, Danvers, MA, USA) protein activation. Oxidative stress was measured by 4-HNE staining (anti-4-hydroxynonenal polyclonal antibody; #ab46545—Abcam, Cambridge, MA, USA) in kidney sections. The intensity of staining was determined using the optical density function of CellSens software (Olympus). Tissue sections were counter-stained with Mayer’s hemalum.

The breast cancer cohort consists of 144 patients diagnosed with invasive breast cancer at Skåne University Hospital, Malmö, Sweden, between 2001 and 2002. The cohort and TMA have previously been described in detail19 (link)52 (link)53 (link). CXCL16 cytoplasmic expression was estimated in the malignant cells (intensity: 0=negative, 1= weak, 2=moderate, 3=strong intensity) and in the stromal fibroblasts (intensity 0=negative-weak, 1=strong intensity). Sections (4 μm thick) of the paraffin embedded tumours were mounted onto glass slides and deparaffinized, prior to antigen retrieval using the PT-link system (DAKO, Glostrup, Denmark) and staining in a Autostainer Plus (DAKO) with the EnVisionFlex High pH-kit (DAKO). All histological sections were counterstained with HE. All primary antibodies used for IHC are shown in Supplementary Table 4 (specificity; clone; dilution; company). Sirius Red staining was performed using in house methods. Cytospins were prepared from monocytes or non-enzymatic cell dissociation buffer-collected (Sigma Aldrich) M2 cultures that were air-dried.